Gibco Cell Culture Hero Webinar: Interrogating cardiac cell states with genetically encoded reporters in hESCs

The adult mammalian heart bears limited regenerative capacity, resulting in the irreversible loss of cardiomyocytes post-cardiac injury, and often culminating in end-stage heart failure. Cardiomyocyte dedifferentiation, a transitional state preceding cell cycle re-entry, represents a required step for cardiomyocyte renewal. By identifying key regulators of both cardiomyocyte differentiation and dedifferentiation, we aim to uncover the regulatory cues driving these dynamic cell state transitions – insights that are essential for advancing cardiac regenerative strategies.

To enable these investigations, we have engineered genomic safe harbour sites in human embryonic stem cells (hESCs), allowing for the efficient and robust integration of transgenes. This approach addresses limitations of lentiviral and piggyBac gene delivery systems, which are prone to transgene silencing during terminal differentiation of cell types. Leveraging on the safe-harbour system, we integrated genetically encoded reporters of dedifferentiation and, alongside a cardiomyocyte-specific MYH6 reporter system, we employed a genome-wide CRISPR loss-of-function screen to uncover essential regulators of cardiomyocyte fate.

Genetically encoded sensors also enable real-time, direct measurement of cellular phenotypes in hESC-derived cardiac cell types, including calcium handling and ATP bioenergetics. Given the heart’s multicellular complexity - comprising cardiomyocytes, fibroblasts, vascular cells, and macrophages – we are extending these tools into 3D culture systems. In particular, the integration of reporter systems into cardiac microtissues provides a more physiologically relevant model to study cellular crosstalk during myocardial stress and injury.

In this talk, we will also explore the integration of genetically encoded reporter systems with 3D cardiac models to enable multimodal readouts, laying the groundwork for next-generation platforms.

Learning Objectives:

• Understand the role of cardiomyocyte dedifferentiation in cardiac regeneration
• Explore genomic engineering strategies used to investigate cardiac cell fate
• Examine the application of genetically encoded sensors in 3D cardiac models